Table of contents

Method of the Year 2013

Nature Methods' choice for Method of the Year 2013 is single-cell sequencing. A collection of articles present the unique considerations related to sequencing single cells and highlight recent applications in biology and medicine. The Methods to Watch feature provides a look at possible future Methods of the Year.


Special Feature



Focus on Method of the Year 2013

Method of the Year 2013 p1


Methods to sequence the DNA and RNA of single cells are poised to transform many areas of biology and medicine.


This Month

Benjamin F. Cravatt p3

Vivien Marx


Building an approach to quantify chinks in a protein's armor.


Research Highlights

Genomes in 3D improve one-dimensional assemblies p5

Chromosome conformation capture data provide scaffolds for de novo genome assemblies.

Unfolding to force pp6 - 7

Applying tiny forces to single molecules at high speed reveals the mechanics behind molecule unfolding.

Electron crystallography goes 3D with MicroED pp6 - 7

Electron diffraction using three-dimensional (3D) crystals may expand the reach of this technique.

Protein nanopores to detect DNA methylation p8

Two groups use nanopore sequencing through a protein pore to detect methylcytosine and hydroxymethylcytosine.

The in vivo RNA structurome p11

Genome-wide, in vivo RNA structure probing helps reveal how RNA structure regulates gene expression.

Methods in Brief

Tools in Brief


News Feature

Focus on Method of the Year 2013

Singled out for sequencing pp13 - 17

Kelly Rae Chi


Single-cell genome and transcriptome sequencing methods are generating a fresh wave of biological insights into development, cancer and neuroscience. Kelly Rae Chi reports.



Focus on Method of the Year 2013

Single-cell sequencing p18

Tal Nawy


A brief overview of how to derive a genome or transcriptome from a single cell.



Focus on Method of the Year 2013

Dissecting genomic diversity, one cell at a time pp19 - 21

Paul C Blainey & Stephen R Quake


Emerging technologies are bringing single-cell genome sequencing into the mainstream; this field has already yielded insights into the genetic architecture and variability between cells that highlight the dynamic nature of the genome.

Focus on Method of the Year 2013

Entering the era of single-cell transcriptomics in biology and medicine pp22 - 24

Rickard Sandberg


Recent technical advances have enabled RNA sequencing (RNA-seq) in single cells. Exploratory studies have already led to insights into the dynamics of differentiation, cellular responses to stimulation and the stochastic nature of transcription. We are entering an era of single-cell transcriptomics that holds promise to substantially impact biology and medicine.

Focus on Method of the Year 2013

The promise of single-cell sequencing pp25 - 27

James Eberwine, Jai-Yoon Sul, Tamas Bartfai & Junhyong Kim


Individual cells of the same phenotype are commonly viewed as identical functional units of a tissue or organ. However, the deep sequencing of DNA and RNA from single cells suggests a more complex ecology of heterogeneous cell states that together produce emergent system-level function. Continuing development of high-content, real-time, multimodal single-cell measurement technologies will lead to the ultimate goal of understanding the function of an individual cell in the context of its microenvironment.


Methods to Watch

Focus on Method of the Year 2013

CRISPRs and epigenome editing p28

Nicole Rusk


Precise alterations to the epigenome with targeted enzymes.

Focus on Method of the Year 2013

Bring on the neuro tools p28

Erika Pastrana


A boost to neuroscience technology development could be transformative.

Focus on Method of the Year 2013

In situ sequencing p29

Tal Nawy


Biologists need methods for sequencing genetic material directly from intact tissues.

Focus on Method of the Year 2013

Tiny tools to measure force p29

Natalie de Souza


Imaging-based sensors are used to map mechanical forces exerted by cells.

Focus on Method of the Year 2013

Single-particle electron cryomicroscopy p30

Allison Doerr


Single-particle electron cryomicroscopy reaches for atomic resolution.

Focus on Method of the Year 2013

Intracellular mini-binders p30

Erika Pastrana


Small, genetically encoded probes for real-time detection and perturbation of cellular events are gaining attention.

Focus on Method of the Year 2013

The power of a crowd p31

Daniel Evanko


For some applications, a crowd of people is superior to the best computational tools available.

Focus on Method of the Year 2013

Self-organizing stem cells p31

Natalie de Souza


Tissue-like organoids with surprisingly complex structures can be formed by stem cells in vitro.


Technology Feature

Pouring over liquid handling pp33 - 38

Vivien Marx


A variety of liquid-handling methods are available for labs large and small. Selecting an approach is not just a matter of budget.


News and Views

The genome shows its sensitive side pp39 - 40

Anil Raj & Graham McVicker


New methods for measuring the sensitivity of chromatin to DNase digestion and Tn5 transposition help us map and interpret the genome's regulatory sequences.

See also: Article by Vierstra et al. | Article by He et al.




Quantitative assessment of single-cell RNA-sequencing methods pp41 - 46

Angela R Wu, Norma F Neff, Tomer Kalisky, Piero Dalerba, Barbara Treutlein, Michael E Rothenberg, Francis M Mburu, Gary L Mantalas, Sopheak Sim, Michael F Clarke & Stephen R Quake


A systematic evaluation of various single-cell RNA-seq approaches reports their sensitivity, accuracy and reproducibility and establishes the high performance of a high-throughput microfluidic method.


Brief Communications

A quantitative liposome microarray to systematically characterize protein-lipid interactions pp47 - 50

Antoine-Emmanuel Saliba, Ivana Vonkova, Stefano Ceschia, Greg M Findlay, Kenji Maeda, Christian Tischer, Samy Deghou, Vera van Noort, Peer Bork, Tony Pawson, Jan Ellenberg & Anne-Claude Gavin


Protein-lipid interactions can be systematically analyzed using a quantitative, multiplexed liposome microarray–based assay (LiMA).

Quantifying RNA allelic ratios by microfluidic multiplex PCR and sequencing pp51 - 54

Rui Zhang, Xin Li, Gokul Ramaswami, Kevin S Smith, Gustavo Turecki, Stephen B Montgomery & Jin Billy Li


For allele-specific expression and RNA editing studies, targeted RNA sequencing using microfluidic multiplexed PCR (mmPCR-seq) gives robust high-throughput measurements of allelic ratios across the dynamic range of gene expression, even for low-quantity or low-quality RNA.

Deciphering laminar-specific neural inputs with line-scanning fMRI pp55 - 58

Xin Yu, Chunqi Qian, Der-yow Chen, Stephen J Dodd & Alan P Koretsky


A line-scanning method is applied to obtain onset times of fMRI responses in rats. The authors show that onset time of the fMRI response can be used to infer information about which cortical layers receive the connectivity input from other brain areas.

HiRIEF LC-MS enables deep proteome coverage and unbiased proteogenomics pp59 - 62

Rui M M Branca, Lukas M Orre, Henrik J Johansson, Viktor Granholm, Mikael Huss, Åsa Pérez-Bercoff, Jenny Forshed, Lukas Käll & Janne Lehtiö


High-resolution isoelectric focusing (HiRIEF) of peptides followed by mass spectrometry analysis, combined with accurate peptide pI prediction, allows a reduction of protein database search space, enabling deep proteome coverage and the discovery of protein-coding loci in human and mouse.

Quantifying the local resolution of cryo-EM density maps pp63 - 65

Alp Kucukelbir, Fred J Sigworth & Hemant D Tagare


A method and software tool, ResMap, for quantifying the local resolution across 3D electron cryo-microscopy density maps is reported.



Coupling transcription factor occupancy to nucleosome architecture with DNase-FLASH pp66 - 72

Jeff Vierstra, Hao Wang, Sam John, Richard Sandstrom & John A Stamatoyannopoulos


By separately sequencing and mapping smaller and larger DNase I fragments from the same DNase I digestion experiment, the approach allows simultaneous profiling of transcription factor footprints relative to nucleosome occupancy.

See also: News and Views by Raj & McVicker

Refined DNase-seq protocol and data analysis reveals intrinsic bias in transcription factor footprint identification pp73 - 78

Housheng Hansen He, Clifford A Meyer, Sheng'en Shawn Hu, Mei-Wei Chen, Chongzhi Zang, Yin Liu, Prakash K Rao, Teng Fei, Han Xu, Henry Long, X Shirley Liu & Myles Brown


Detailed analysis of DNase-seq protocols reveals the importance of choosing the right enzyme concentration and fragment length and cautions that many transcription factor footprints may represent cutting bias.

See also: News and Views by Raj & McVicker

A chemoproteomic platform to quantitatively map targets of lipid-derived electrophiles pp79 - 85

Chu Wang, Eranthie Weerapana, Megan M Blewett & Benjamin F Cravatt


A competitive activity–based protein profiling method is reported for quantifying the reactivity of lipid-derived electrophilic compounds with cysteine residues in the human proteome.

Parallel measurement of dynamic changes in translation rates in single cells pp86 - 93

Kyuho Han, Ariel Jaimovich, Gautam Dey, Davide Ruggero, Oded Meyuhas, Nahum Sonenberg & Tobias Meyer


A system to monitor translation regulation in living cells is reported. By fusing a fluorescent reporter that has a controllable destabilization domain to translation regulatory motifs, the authors analyze the contribution of these motifs to changes in translation in individual cells under different experimental situations.

Integrating protein-protein interaction networks with phenotypes reveals signs of interactions pp94 - 99

Arunachalam Vinayagam, Jonathan Zirin, Charles Roesel, Yanhui Hu, Bahar Yilmazel, Anastasia A Samsonova, Ralph A Neumüller, Stephanie E Mohr & Norbert Perrimon


An approach is presented for predicting the nature of the relationship (activating or inhibiting) between interacting proteins via integration of phenotypic information with protein-protein interaction networks.

A holidic medium for Drosophila melanogaster pp100 - 105

Matthew D W Piper, Eric Blanc, Ricardo Leitão-Gonçalves, Mingyao Yang, Xiaoli He, Nancy J Linford, Matthew P Hoddinott, Corinna Hopfen, George A Soultoukis, Christine Niemeyer, Fiona Kerr, Scott D Pletcher, Carlos Ribeiro & Linda Partridge


A chemically defined diet for Drosophila melanogaster is described. It should enable a variety of behavioral, metabolic and fitness studies where controlled nutrition is important.

Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny pp106 - 112

Xiaolei Yin, Henner F Farin, Johan H van Es, Hans Clevers, Robert Langer & Jeffrey M Karp


This paper reports culture conditions for the expansion of near-homogeneous populations of mouse Lgr5+ intestinal stem cells. These methods will enable the study of intestinal biology and potentially that of other tissues.